1. Technical Field
The present invention relates to an antibiotic-independent vector for constant high-expression. More particularly, the invention relates to a high-expression vector in which a recombinant gene can be stably transcribed and highly expressed, since antibiotic-resistant genes, such as ampicillin and the like, are not utilized, while genes essential for cell growth and proliferation are adopted as a selective marker to secure a stable gene expression system.
2. Background Art
To manufacture useful proteins using a genetic engineering technique, an expression system is used that exploits a proper promoter and host cell established in its culture protocol. Precisely, microbes, such as Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae etc., animal cells, insect cells, and plant cells can all be selected as host cells. In addition, to replicate plasmids consistently, an antibiotic-resistant gene is inserted into the cloning vector, which is then fermented based on the addition of antibiotics into the culture medium or a proper inducer for the high-expression of the target gene.
Generally, ampicillin, kanamycin, tetracycline, or the like are adopted as a selective marker during the fermentation to perform the gene cloning and produce recombinant enzymes for industrial uses and recombinant proteins for medical purposes. However, such antibiotic-resistant genes are apt to lose their activity over time. Therefore, in a continuous fermentation process, the cultured product of the host cells is degraded or diluted as the number of generations increases. The antibiotics added into the culture medium decompose, the stability of the highly sensitive plasmids decreases, and thus the amount of proteins expressed from the cloned exogenous genes also decreases. In the case that the protein is a kind of food additive, the antibiotics added for the cultivation must be removed before proceeding to the expression. This also results in high costs for gene production on a large scale, as inducers such as isopropyl-β-D-thiogalactoside(IPTG) are expensive.
Accordingly, many studies have already attempted to overcome the foregoing and other disadvantages. Concretely, Korean Patent Registration No. 0231919 discloses an expression vector that exploits the temperature induction of a phage λ promoter instead of the expensive IPTG and includes the application of an antibiotic-resistant gene. However, when induction methods are used that involve temperature without inducers, temperature increases are hard to manage optimally. Also, the expression ratio is lower than that in conventional methods using inducers. U.S. Pat. No. 5,919,663 illustrates that the modified pur A vector (adenylosuccinate synthetase) can be applied to maintain a stable plasmid without the use of an antibiotic-resistant gene. Presently, auxotrophic strains are used to minimize the production of amino acids and by-products inhibiting the synthesis of target amino acids. However, if mutant strains are deficient as synthetic genes in the amino acids essential for cell growth, a refined medium rather than a complex one is required in which the L-amino acid content has been adjusted, namely the composition of the medium is quantified.
Therefore, a novel gene expression system is required that facilitates stable gene production, reduces costs, and provides excellent industrial application.